In a diverse group of organisms ranging from plants to vertebrates, double-stranded RNA can silence gene expression in a sequence specific manner. We have made substantial progress in deciphering the mechanism underlying this phenomenon which has been termed variously RNA interference (RNAi) or post-transcriptional gene silencing. In this application, we propose to develop RNAi as a tool for analyzing gene function in mammalian cells. Recent data has suggested that dsRNA can suppress gene expression in Chinese Hamster Ovary cells. We will use approaches similar to those which we have previously used in Drosophila to determine whether this effect occurs through and RNAi mechanism. If so, we propose to develop tools which will allow the use of RNAi in CHO cells for probing gene function. Using CHO cells as a starting point, and incorporating our ever-advancing understanding of the RNAi mechanism, our ultimate goal is to reconstitute the RNAi machinery in a broad range of mammalian cell types. In this Phase I application, we will test whether ectopic expression of components of the RNAi machinery that we have already identified can convert cells to an RNAi competent state. Furthermore, we will develop a strategy for executing an unbiased screen for activation of RNAi in mammalian cells. PROPOSED COMMERCIAL APPLICATION: With accumulating genomic information, novel technologies are required to speed analysis of gene function in the post-genomic era. While approaches for creating loss-of-function mutations in mammals do exist, they are cumbersome, time consuming and technically difficult. The development of facile gene silencing methodology for cultured mammalian cells and animals would revolutionize biology.